Tomato plant line TZ368

ABSTRACT

The present invention relates to a new and distinct inbred tomato lines and hybrids. This invention also relates to plants and seeds of such inbred tomato lines and hybrids, and to parts thereof. The invention also relates to methods for producing a tomato plant produced by crossing such inbred tomato lines and hybrids with themselves or other tomato plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/643,619,now U.S. Pat. No. 7,612,261, filed Dec. 22, 2006 which claims thebenefit of European Union Community Plant Variety Right No. 2006/0353,filed Feb. 2, 2006 under 35 U.S.C. 119(f). The aforementionedapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, and to newand distinct inbred lines and hybrids of tomato (Lycopersiconesculentum), and to method of making and using such inbred lines andhybrids.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to develop new, unique and superiorcultivars. Theoretically, a breeder can generate billions of differentgenetic combinations via crossing, selfing and selection. A breeder hasno direct control at the cellular level. Therefore, two breeders willnever develop the same line, or even very similar lines, havingprecisely the same traits. Descriptions of breeding methods that arecommonly used for different traits and crops, as well as specificallyfor tomato, can be found in one of several reference books (e.g.,Allard, R. W. (1960) Principles of Plant Breeding; Simmonds, N. W.(1979) Principles of Crop Improvement; Sneep, J. et al., (1979) TomatoBreeding (p. 135-171) in: Breeding of Vegetable Crops, Mark J. Basset,(1986, editor), The Tomato crop: a scientific basis for improvement, byAtherton, J. G. & J. Rudich, (1986, editors); Plant BreedingPerspectives; Fehr, (1987) Principles of Cultivar Development—Theory andTechnique).

The method chosen for breeding or selection depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thecultivar (i.e. variety) used commercially (e.g. F1 hybrid, or anopen-pollinated variety). The complexity of the inheritance influencesthe choice of breeding method. One simple method of identifying asuperior plant is to observe its performance relative to otherexperimental plants or to a widely grown standard cultivar, and toobserve its performance in hybrid combinations with other plants. Ifsingle observations are inconclusive for establishing distinctness,observations in multiple locations and seasons provide a better estimateof its genetic worth. Proper testing and evaluation should detect anymajor faults and establish the level of superiority or improvement overcurrent cultivars.

The development of commercial tomato hybrids requires the development ofhomozygous inbred parental lines. In breeding programs desirable traitsfrom two or more germplasm sources or gene pools are combined to developsuperior breeding lines. Desirable inbred or parent lines are developedby continuous selfing and selection of the best breeding lines,sometimes utilizing molecular markers to speed up the selection process.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be produced indefinitely, as long as thehomogeneity and the homozygosity of the inbred parents is maintained. Asingle-cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. Much of the hybrid vigor exhibited by F1 hybridsis lost in the next generation (F2). Consequently, seed harvested fromhybrid varieties is not used for planting stock.

There are numerous steps involved in the breeding and development of anynew and novel, desirable plant germplasm with superior combiningability. Plant breeding begins with the analysis and definition ofproblems and weaknesses of the current germplasm, the establishment ofprogram goals, and definition of specific breeding objectives. The nextstep is selection of germplasm that possess the traits to meet theprogram goals and the definition of the best breeding method to reachthose goals. The objective is to combine in a single hybrid variety animproved combination of desirable traits from the parental germplasm.Important characteristics may include higher yield, better flavor,improved color and field holding ability, resistance to diseases andinsects, tolerance to drought and heat, along with characteristicsrelated to hybrid seed yields to lower the cost of hybrid seedproduction.

Tomato is a very important crop in all continents of the world. Severalplant species associated with the Solanum group have been familiar tomankind since ancient times, and are of great agricultural importance.Solanum species have a general adaptation to variable climatic growingconditions. Tomato (Lycopersicon esculentum L.) belongs to theSolaneaceous family. All varieties in the species esculentum areself-pollinating. Most other species in the genus Lycopersicon arecross-pollinating. Cross-pollination is affected by insect vectors, mostcommonly by the honey- or bumblebees. Tomato, like most otherLycopersicon species, is highly variable. Variability in populations isdesired for wide adaptation and survival. Tomato is adapted to warmsummer growing conditions, but can also be grown in heated greenhousesunder winter conditions. The introduction of hybrid cultivars in the1950's provided a magnitude of benefits like increased yield, betterholding ability, adaptation to expanded growing seasons through the useof protected cultivation and improved disease resistance, which resultedin large-scale production of tomato as a commercial crop.

The goal in tomato breeding is to make continued improvements in hybridtomato yields, in other horticultural characteristics, as well as inquality traits, in order to meet continuous demands for better tomatocultivars in different growing regions of the world.

SUMMARY OF THE INVENTION

The present invention discloses new and distinct inbred tomato lines andhybrids of tomato (Lycopersicon esculentum). The present invention alsodiscloses methods of making and using such inbred lines and hybrids.

In one embodiment, the present invention discloses a new and distinctinbred tomato line, designated TZ367. In one embodiment, the presentinvention relates to a new and distinct inbred tomato line, designatedTZ368. This invention also discloses seeds of inbred tomato lines TZ367and TZ368, plants of inbred tomato lines TZ367 and TZ368, and parts ofsaid plants, such as pollen, ovule or fruit. The present invention alsodiscloses methods for producing a tomato plant produced by crossing aplant of inbred line TZ367 or TZ368 with itself or another tomato line.

This invention also relates to methods for producing other inbred tomatolines derived from inbred tomato line TZ367 or TZ368, and to the inbredtomato lines derived by the use of those methods. This invention furtherrelates to hybrid tomato seeds and plants produced by crossing inbredtomato line TZ367 or TZ368 with another tomato line. This inventionfurther relates to hybrid tomato seeds and plants produced by crossinginbred tomato line TZ367 with inbred tomato line TZ368.

In one embodiment, this invention also discloses seeds of tomato hybridSX 387, plants of tomato hybrid SX 387, and parts of said plants, suchas pollen, ovule or fruit. The present invention also discloses methodsfor producing a tomato plant comprising crossing tomato hybrid SX 387with itself or another tomato line.

The invention further discloses method of producing seed of a plant ofthe present invention comprising crossing an inbred line or hybrid ofthe present invention with itself or with another line or hybrid, andseed produced by such method. The invention also discloses methods ofvegetatively propagating a plant of the present invention, and to plantsproduced by such methods. This invention also methods for producing afruit of a tomato plant of the present invention and to fruits producedby such methods.

A tomato plant of the invention may further comprise a cytoplasmicfactor or other factor that is capable of conferring male sterility.Male sterility may also be provided by nuclear genes such as therecessive ms gene.

In another aspect, the present invention provides regenerable cells foruse in tissue culture. The tissue culture will preferably be capable ofregenerating plants having the physiological and morphologicalcharacteristics of the foregoing tomato plants, and of regeneratingplants having substantially the same genotype as the foregoing tomatoplants. Preferably, the regenerable cells in such tissue cultures willbe embryos, protoplasts, meristematic cells, callus, pollen, leaves,anthers, stems, petioles, roots, root tips, fruits, seeds, flowers,cotyledons, hypocotyls or the like. Still further, the present inventionprovides tomato plants regenerated from the tissue cultures of theinvention.

In another aspect, the present invention provides for single geneconverted plants of inbred tomato lines TZ367 and TZ368, or hybridSX387. The single transferred gene may preferably be a dominant orrecessive allele. Preferably, the single transferred gene will confersuch trait as male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility,improved harvest characteristics, enhanced nutritional quality, improvedprocessing characteristics. The single gene may be a naturally occurringtomato gene or a transgene introduced through genetic engineeringtechniques. The present invention also discloses methods for producing atomato plant containing in its genetic material one or more transgenesand to the transgenic tomato plants produced by that method. Theinvention further provides methods for developing tomato plant in atomato plant breeding program using plant breeding technique includingrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection and transformation. Seeds, tomato plant, and parties thereofproduced by such breeding methods are also part of the invention.

DEFINITIONS

In the description and tables that follow, a number of terms are used.The terms are used to provide a clear understanding of thespecifications and are used in accordance with the terminology definedin the UPOV Technical Guidelines for tomato (TG/4417), which isincorporated herein by reference in its entirety. The followingdefinitions are also provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes. Backcrossing. Backcrossing isa process in which a breeder repeatedly crosses hybrid progeny back toone of the parents, for example, a first generation hybrid F1 with oneof the parental genotype of the F1 hybrid.

Regeneration. Regeneration refers to the development of a plant fromtissue culture. Single gene converted. Single gene converted orconversion plant refers to plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of an inbred arerecovered in addition to the single gene transferred into the inbred viathe backcrossing technique or via genetic engineering.

Soluble Solids. Soluble solids refers to the percent of solid materialfound in the fruit tissue, the vast majority of which is sugars. Solublesolids are directly related to finished processed product yield ofpastes and sauces. Soluble solids are estimated with a refractometer,and measured as degrees brix.

ph: the pH is a measure of acidity.

Viscosity: the viscosity or consistency of tomato products is affectedby the degree of concentration of the tomato, the amount of and extentof degradation of pectine, the size, shape and quality of the pulp, andprobably to a lesser extent, by the proteins, sugars and other solubleconstituents. The viscosity is measured in Bostwick centimeters by usinginstruments such as a Bostwick Consistometer.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Predicted paste bostwick: the predicted paste bostwick is the flowdistance of tomato paste diluted to 12 degrees brix and heated prior toevaluation. Dilution to 12 degrees brix for bostwick measurement is astandard method used by industry to evaluate product consistency. Thelower the number, the thicker the product and therefore more desirablein consistency oriented products such as catsup. The following formulais usually used to evaluate the predicted paste bostwick: Predictedpaste bostwick=−1.53+(1.64*juice brix)+(0.5*juice bostwick)

Determinate tomatoes: varieties that come to fruit all at once, thenstop bearing. They are best suited for commercial growing since they canbe harvested all at once.

Relative maturity: relative maturity is an indication of time until atomato genotype is ready for harvest. A genotype is ready for harvestwhen 90% or more of the tomatoes are ripe.

Semi-erect habit: a semi-erect plant has a combination of lateral andupright branching and has an intermediate type habit between a prostateplant habit, having laterally growing branching with fruits most of thetime on the ground and an erect plant habit with branching goingstraight up with fruit being off the ground.

Deep globe shape: a tomato fruit being slightly wider than longer butstill having a round shape.

Flesh color: the color of the tomato flesh that can range fromorange-red to dark red when at ripe stage (harvest maturity).

Uniform ripening: a tomato that ripens uniformly, i.e., that has nogreen discoloration on the shoulders. The uniform ripening is controlledby a single recessive gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses new and distinct inbred tomato lines andhybrids of tomato (Lycopersicon esculentum). The present invention alsodiscloses methods of making and using such inbred lines and hybrids. Inone embodiment, the present invention discloses a new and distinctinbred tomato line, designated TZ367. In one embodiment, the presentinvention relates to a new and distinct inbred tomato line, designatedTZ368. In one embodiment, this invention also discloses tomato hybrid SX387.

In one embodiment, the tomato inbred lines and hybrids of the presentinvention are capable of producing a fruit, which has a sweet taste andhas a brown-red color. Typically, the fruit of a tomato plant of theinstant invention comprises about 3.7 g to about 4.0 g total sugars(i.e. glucose, fructose and sucrose) per 100 g fresh weight and about400 mg to about 550 mg citric acid per 100 g fresh weight. However it isunderstood that these values are dependent on the environmentalconditions under which a tomato plant is grown, and may accordinglyvary. Additional characteristics of the tomato inbred lines and hybridof the instant invention are shown in Tables 1 to 3 below.

The tomato inbred lines and hybrid of the instant invention have shownuniformity and stability for all traits. The inbred lines of the presentinvention have been self-pollinated and planted for a sufficient numberof generations, with careful attention to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected. In one embodiment, the present inventiondiscloses a method of producing seed of a tomato plant of the presentinvention comprising: a) growing a plant of the present invention; b)allowing said plant to self-pollinate; c) harvesting seeds from saidplant.

The inbred lines of the instant invention have superior characteristics,and provide excellent parental lines in crosses for producing firstgeneration (F1) hybrid tomato. In one embodiment, the present inventionalso discloses a method of producing a hybrid tomato seed. In oneembodiment, the method comprises crossing a plant of an inbred tomatoline of the instant invention with a plant of another tomato line. Inone embodiment, inbred line TZ367 is crossed with inbred line TZ368 toobtain hybrid SX 387. In one embodiment, inbred lines TZ367 and TZ368are used are male parent or female parent to obtain hybrid SX 387.

Great care is taken during hybrid seed production to preventcontamination of lots of hybrid seeds with seeds of parent inbred lines,in particular of seeds of the female parent. During the production ofthe hybrid seed, care is taken to harvest only seeds produced byflowers, which have been cross-pollinated by the pollen of the maleparent, while avoiding seeds produced by flowers, which have beenself-pollinated. After harvest, grow-out tests are typically conductedto test for the undesired presence of seeds of the parental lines byobserving the phenotypic characteristics of the hybrids andcorresponding parents. Purity test are also conducted using biochemicaland molecular markers. Lots of hybrids seeds, which do not producesatisfying results are not released.

A tomato plant can also be propagated vegetatively. A part of the plant,for example a shoot tissue, is collected, and a new plant is obtainedfrom the part. Such part typically comprises an apical meristem of theplant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots, or is grafted onto a tomato plant or a rootstock prepared tosupport growth of shoot tissue. This is achieved using methodswell-known in the art. Accordingly, in one embodiment, a method ofvegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, a fruit isharvested from said plant.

The present invention also contemplates a tomato plant regenerated froma tissue culture of an inbred or hybrid plant of the present invention.As is well known in the art, tissue culture of tomato can be used forthe in vitro regeneration of a tomato plant. Kartha, K. K., Gamborg, O.L., Shyluk, J. P., and Constabel, F., Morphogenetic investigations on invitro leaf cultures of tomato (Lycopersicon esculentum Mill. cv.Starfire) and high frequency plant regeneration, Z. Pflanzenphysiol.,77, 292, 1976.

In one embodiment, the present invention discloses a method of producinga tomato fruit. In one embodiment, such method comprises growing a plantof the instant invention to produce a tomato fruit, and harvesting saidtomato fruit. In one embodiment, the method further comprises packingsaid fruit in a suitable container. In one embodiment, the methodfurther comprises shipping said fruit. In one embodiment, a fruit of atomato plant of the present invention is used in fresh consumption or isprocessed.

Tables 1 to 3 below disclose additional characteristics of the tomatoplants of the present invention.

TABLE 1 Characteristics of inbred tomato line TZ367 Tomato plants weregrown in plastic greenhouses in Almeria, Spain under standardconditions. Characteristics Seedling: anthocyanin coloration ofhypocotyl 1 absent/9 present 9 Stem: type 1 very rigid/2 flexible. 2Stem: Pubescence 1 absent/3 few/5 medium/7 strong. 5 Stem: Number ofleaves under the first inflorescence 3 few/5 medium/7 many. 7 Stem:Internode length (between the 1st and 3 rd inflorescence 3 short/5medium/7 long. 5 Plant: growth type 1 determinate/2 indeterminate/3semi-determinate 2 Plant: height 1 very low/3 low/5 medium/7 high/9 veryhigh. 5 Plant: vigour 3 weak/5 medium/7 strong 5 Plant: speed of growth(Indeterminant varieties only) 1 very slow/3 slow/5 medium/7 fast/9 veryfast 5 Leaf: pose/attitude 3 semi upright/5 horizontal/7 downwards. 7Leaf: length 3 short/5 medium/7 long. 5 Leaf: width 3 narrow/5 medium/7wide. 5 Leaf: density of the foliage 3 weak/5 medium/7 strong 5 Leaf:division of blade (see drawings on Instructions tab) 1 pinnate/9bipinnate 9 Leaf: type (see drawings on Instructions tab) 1 type1/2type2/3 type3/4 type4 1 Leaf: intensity of green colour 1 very light/3light/5 medium/7 dark/9 very dark 7 Leaf: anthocyanin coloration of theveins 1 absent/9 present. 9 Inflorescence: type 1 single flowering/2multiflowering. 2 Peduncle: abscission layer (see drawing onInstructions tab) 1 absent (jointless)/9 present (jointed) 9 Flower:fasciation (1 st flower of inflorescence) 1 absent/9 present. 9 Flower:pubescence of style 1 absent/9 present. 9 Flower: color 1 yellow/2orange. 1 Fruit: ribbing at stem end (calyx) 1 absent or very weak/2weak/3 strong 4 very strong 2 Fruit: size 1 very small/3 small/5medium/7 large/9 very large (130gram) 3 Fruit: shape in longitudinalsection 1 flattened/2 slightly flattened/3 round/4 rectangular/5 3

 

 

 

 

 

 

 

cylindrical/6 heart-shaped/7 obovoid/8 ovoid/9 pear- shaped/10 stronglypear-shaped Fruit: shape in longitudinal section (Spanishclassification) 1 heartshaped/3 elliptic/5 cylindrical/7 pyramidshaped.3 Fruit: shape in transverse section 1 round/2 angular/3 irregular. 1Fruit: ratio size/height 1 very low/3 low/5 medium/7 high/9 very high. 5Fruit: length of pedicel (from abscission layer to calyx) 3 short/7long. 5 Fruit: pedicel area 1 smooth/3 little globe/5 medium globe/7high globe. 3 Fruit: size of pedicel scar 3 small/5 medium/7 big. 3Fruit: shape of peduncular part 1 smooth/5 slightly ribbed/9 ribbed. 3Fruit: size of corky area around pedicel scar 3 small/5 medium/7 big. 3Fruit: shape of pistil scar 1 pointed/2 starshaped/3 striped/4irregular. 2 Fruit: predominant number of locules 1 two/2 two andthree/3 three and four/4 four, five, six/ 2 5 more then six Fruit:predominant number of locules (Spanish classification) 2 mainlytwo/3mainly trhee/4 mainly four/5 mainly five/6 mainly 4 six or moreFruit: shape at blossum end (see drawings on Instructions tab) 1 veryindented/3 indented/5 round/7 pointed/9 extreme 5 pointed. Fruit: sizeof core (in cross-section) 3 small/7 big. 3 Fruit: green shoulder(before maturity) 1 absent/9 present 9 Fruit: intensity of greenshoulder before maturity. 1 absent/3 weak/5 medium/7 strong 9 verystrong. 9 Fruit: thickness of pericarp 3 thin/5 medium/7 thick. 5 Fruit:color before maturity 3 lightgreen/5 mediumgreen/7 darkgreen. 7 Fruit:color at maturity 1 yellow/2 orange/3 pink/4 red/5 darkred/6 brownred. 6Fruit: color of the epidermis at maturity 1 colorless/2 yellow. 2 Fruit:color of flesh (at maturity) 1 yellow/2 orange/3 pink/4 red 4 Fruit:homogenity of size 1 heterogene/9 homogene 9 Fruit: firmness 1 verysoft/3 soft/5 medium/7 firm/9 very firm. 6 Time of flowering (if grownin the greenhouse, to be observed on 3 early/5 medium/7 late. 7 3 rdflower of the 2nd truss) Time of maturity 1 very early/3 early/5medium/7 late/9 very late 5 Additional information Resistances to pestsand diseases: Expression of silvering 0 not tested/1 absent (tol. tosilvering)/8 present (susc. to 0 silvering) Meloidogyne incognita 1absent/9 present 1 Verticillium race 0 1 absent/9 present 9 Fusariumoxysporum f. sp. lycopersici race 0 (old 1) 1 absent/9 present 9Fusarium oxysporum f. sp. lycopersici race 1 (old 2) 1 absent/9 present9 Fusarium oxysporium f. sp. radicis lycopersici 0 not tested/1 absent/9present. 1 Cladosporium fulvum group 0 (Indeterminate varieties only) 0not tested/1 absent/9 present. 1 Cladosporium fulvum group A(Indeterminate varieties only) 1 absent/9 present 1 Cladosporium fulvumgroup B (Indeterminate varieties only) 1 absent/9 present 1 Cladosporiumfulvum group C (Indeterminate varieties only) 1 absent/9 present 1Cladosporium fulvum group D (Indeterminate varieties only) 1 absent/9present 1 Cladosporium fulvum group E (Indeterminate varieties only) 1absent/9 present 1 TMV race 0 0 not tested/1 absent/9 present 9 Tomatomosaic virus (ToMV) strain 0 1 absent/9 present 9 Tomato mosaic virus(ToMV) strain 1 1 absent/9 present 9 Tomato mosaic virus (ToMV) strain 21 absent/9 present 9 Tomato mosaic virus (ToMV) strain 1-2 0 nottested/1 absent/9 present 9 Allele Tm 1 Tm1/2 Tm2/3 Tm22 3 Specialconditions for the examination of the variety Type of culture: 1glasshouse/2 outdoor/3 other . . . 1 Type of culture: 1 staked/2 semistaked/3 not staked 1 Main use: 1 fresh market or garden/2 industrialprocessing 1 If 1 fresh market or garden: 1 single/2 truss/3 1 other, .. .

TABLE 2 Characteristics of inbred tomato line TZ368 Tomato plants weregrown in plastic greenhouses in Almeria, Spain under standardconditions. Characteristics Seedling: anthocyanin coloration ofhypocotyl 1 absent/9 present 9 Stem: type 1 very rigid/2 flexible. 1Stem: Pubescence 1 absent/3 few/5 medium/7 strong. 5 Stem: Number ofleaves under the first inflorescence 3 few/5 medium/7 many. 6 Stem:Internode length (between the 1st and 3 rd inflorescence 3 short/5medium/7 long. 6 Plant: growth type 1 determinate/2 indeterminate/3semi-determinate 2 Plant: height 1 very low/3 low/5 medium/7 high/9 veryhigh. 7 Plant: vigour 3 weak/5 medium/7 strong 5 Plant: speed of growth(Indeterminant varieties only) 1 very slow/3 slow/5 medium/7 fast/9 veryfast 7 Leaf: pose/attitude 3 semi upright/5 horizontal/7 downwards. 5Leaf: length 3 short/5 medium/7 long. 3 Leaf: width 3 narrow/5 medium/7wide. 3 Leaf: density of the foliage 3 weak/5 medium/7 strong 5 Leaf:division of blade (see drawings on Instructions tab) 1 pinnate/9bipinnate 9 Leaf: type (see drawings on Instructions tab) 1 type1/2type2/3 type3/4 type4 1 Leaf: intensity of green colour 1 very light/3light/5 medium/7 dark/9 very dark 5 Leaf: anthocyanin coloration of theveins 1 absent/9 present. 9 Inflorescence: type 1 single flowering/2multiflowering. 2 Peduncle: abscission layer (see drawing onInstructions tab) 1 absent (jointless)/9 present (jointed) 9 Flower:fasciation (1 st flower of inflorescence) 1 absent/9 present. 1 Flower:pubescence of style 1 absent/9 present. 9 Flower: color 1 yellow/2orange. 1 Fruit: ribbing at stem end (calyx) 1 absent or very weak/2weak/3 strong/4 very strong 1 Fruit: size 1 very small/3 small/5medium/7 large/9 very large (80 gram) 3 Fruit: shape in longitudinalsection 1 flattened/2 slightly flattened/3 round/4 rectangular/5 3

 

 

 

 

 

 

 

cylindrical/6 heart-shaped/7 obovoid/8 ovoid/9 pear- shaped/10 stronglypear-shaped Fruit: shape in longitudinal section (Spanishclassification) 1 heartshaped/3 elliptic/5 cylindrical/7 pyramidshaped.3 Fruit: shape in transverse section 1 round/2 angular/3 irregular. 1Fruit: ratio size/height 1 very low/3 low/5 medium/7 high/9 very high. 5Fruit: length of pedicel (from abscission layer to calyx) 3 short/7long. 5 Fruit: pedicel area 1 smooth/3 little globe/5 medium globe/7high globe. 1 Fruit: size of pedicel scar 3 small/5 medium/7 big. 3Fruit: shape of peduncular part 1 smooth/5 slightly ribbed/9 ribbed. 1Fruit: size of corky area around pedicel scar 3 small/5 medium/7 big. 3Fruit: shape of pistil scar 1 pointed/2 starshaped/3 striped/4irregular. 2 Fruit: predominant number of locules 1 two/2 two andthree/3 three and four/4 four, five, six/ 4 5 more then six Fruit:predominant number of locules (Spanish classification) 2 mainly two/3mainly trhee/4 mainly four/5 mainly five/6 mainly 3 six or more Fruit:shape at blossum end (see drawings on Instructions tab) 1 veryindented/3 indented/5 round/7 pointed/9 3 extreme pointed. Fruit: sizeof core (in cross-section) 3 small/7 big. 3 Fruit: green shoulder(before maturity) 1 absent/9 present 9 Fruit: intensity of greenshoulder before maturity. 1 absent/3 weak/5 medium/7 strong/9 verystrong. 9 Fruit: thickness of pericarp 3 thin/5 medium/7 thick. 5 Fruit:color before maturity 3 lightgreen/5 mediumgreen/7 darkgreen. 5 Fruit:color at maturity 1 yellow/2 orange/3 pink/4 red/5 darkred/6 brownred. 6Fruit: color of the epidermis at maturity 1 colorless/2 yellow. 2 Fruit:color of flesh (at maturity) 1 yellow/2 orange/3 pink/4 red 4 Fruit:homogenity of size 1 heterogene/9 homogene 9 Fruit: firmness 1 verysoft/3 soft/5 medium/7 firm/9 very firm. 7 Time of flowering (if grownin the greenhouse, to be observed 3 early/5 medium/7 late. 5 on 3 rdflower of the 2nd truss) Time of maturity 1 very early/3 early/5medium/7 late/9 very late 5 Additional information Resistances to pestsand diseases: Expression of silvering 0 not tested/1 absent (tol. tosilvering)/8 present (susc. 0 to silvering) Meloidogyne incognita 1absent/9 present 9 Verticillium race 0 1 absent/9 present 9 Fusariumoxysporum f. sp. lycopersici race 0 (old 1) 1 absent/9 present 9Fusarium oxysporum f. sp. lycopersici race 1 (old 2) 1 absent/9 present9 Fusarium oxysporium f. sp. radicis lycopersici 0 not tested/1 absent/9present. 1 Cladosporium fulvum group 0 (Indeterminate varieties only) 0not tested/1 absent/9 present. 1 Cladosporium fulvum group A(Indeterminate varieties only) 1 absent/9 present 1 Cladosporium fulvumgroup B (Indeterminate varieties only) 1 absent/9 present 1 Cladosporiumfulvum group C (Indeterminate varieties only) 1 absent/9 present 1Cladosporium fulvum group D (Indeterminate varieties only) 1 absent/9present 1 Cladosporium fulvum group E (Indeterminate varieties only) 1absent/9 present 1 TMV race 0 0 not tested/1 absent/9 present 1 Tomatomosaic virus (ToMV) strain 0 1 absent/9 present 1 Tomato mosaic virus(ToMV) strain 1 1 absent/9 present 1 Tomato mosaic virus (ToMV) strain 21 absent/9 present 1 Tomato mosaic virus (ToMV) strain 1-2 0 nottested/1 absent/9 present 1 Stemphylium spp. 0 not tested/1 absent/9present 1 Special conditions for the examination of the variety Type ofculture: 1 glasshouse/2 outdoor/3 other . . . 1 Type of culture 1staked/2 semi staked/3 not staked 1 Main use: 1 fresh market or garden/2industrial processing 1 If 1 fresh market or garden: 1 single/2 truss/31 other, . . .

TABLE 3 Characteristics of tomato hybrid SX 387 Tomato plants were grownin plastic greenhouses in Almeria, Spain under standard conditions.Characteristics Seedling: anthocyanin coloration of hypocotyl 1 absent/9present 9 Stem: type 1 very rough/2 flexible. 21 Stem: Pubescence 1absent/3 few/5 medium/7 strong. 5 Stem: Number of leaves under the firstinflorescence 3 few/5 medium/7 many. 7 Stem: Internode length (betweenthe 1st and 3 rd inflorescence 3 short/5 medium/7 long. 6 Plant: growthtype 1 determinate/2 indeterminate/3 semi-determinate 2 Plant: height 1very low/3 low/5 medium/7 high/9 very high. 6 Plant: speed of growth(Indeterminant varieties only) 1 very slow/3 slow/5 medium/7 fast/9 veryfast 6 Leaf: pose 3 semi upright/5 vertical/7 downwards. 7 Leaf: length3 short/5 medium/7 long. 5 Leaf: width 3 narrow/5 medium/7 wide. 5 Leaf:division of blade 1 pinnate/9 bipinnate 9 Leaf: type (see drawings) 1type1/2 type2/3 type3/4 type4 1 Leaf: intensity of green colour 1 verylight/3 light/5 medium/7 dark/9 very dark 7 Leaf: anthocyanin colorationof the veins 1 absent/9 present. 9 Inflorescence: type 1 singleflowering/2 multiflowering. 2 Peduncle: abscission layer 1 absent(jointless)/9 present (jointed) 9 Flower: fasciation (1 st flower ofinflorescence) 1 absent/9 present. 9 Flower: pubescence of style 1absent/9 present. 9 Flower: color 1 yellow/2 orange. 1 Fruit: ribbing atstem end 1 absent or very weak/2 weak/3 strong/4 very strong 2 Fruit:size 1 very small/3 small/5 medium/7 large/9 very large (140 gram) 5Fruit: ribbing at calyx end 1 absent/3 few/5 medium/7 strong. 3 Fruit:shape in longitudinal section 1 flattened/2 slightly flattened/3 round/4rectangular/5 2 cylindrical/6 heart-shaped/7 obovoid/8 ovoid/9pear-shaped/10 strongly pear-shaped Fruit: shape in transverse section 1round/2 angular/3 irregular. 1 Fruit: ratio height/size 1 very low/3low/5 medium/7 high/9 very high. 3 Fruit: length of pedicel (fromabscission layer to calyx) 3 short/7 long. 3 Fruit: pedicel area 1smooth/3 little globe/5 medium globe/7 high globe. 3 Fruit: size ofpedicel scar 3 small/5 medium/7 big. 4 Fruit: size of corky area aroundpedicel scar 3 small/5 medium/7 big. 3 Fruit: shape of pitil scar 1pointed/2 starshaped/3 striped/4 irregular. 2 Fruit: predominant numberof locules 1 two/2 two and three/3 three and four/4 four, five, six/5more 3 than six Fruit: shape at blossum end 1 very indented/3 indented/5round/7 pointed/9 extreme pointed. 3 Fruit: size of core (incross-section) 3 small/7 big. 3 Fruit: green shoulder (before maturity)1 absent/9 present 9 Fruit: intensity of green shoulder before maturity.1 absent/3 weak/5 medium/7 strong 9 very strong. 9 Fruit: thickness ofpericarp 3 thin/5 medium/7 thick. 5 Fruit: color before maturity 3lightgreen/5 mediumgreen/7 darkgreen. 7 Fruit: color at maturity 1yellow/2 orange/3 pink/4 red/5 darkred/6 brownred. 6 Fruit: color of theepidermis at maturity 1 colorless/2 yellow. 2 Fruit: color of flesh (atmaturity) 1 yellow/2 orange/3 pink/4 red 4 Fruit: firmness 1 very soft/3soft/5 medium/7 firm/9 very firm. 7 Time of flowering (if grown in theopenfield, to be observed on 3 early/5 medium/7 late. 3 rd flower of the2nd truss) Time of flowering (if grown in the greenhouse, to be observedon 3 early/5 medium/7 late. 5 3 rd flower of the 2nd truss) Time ofmaturity 1 very early/3 early/5 medium/7 late/9 very late 6 Additionalinformation Resistances to pests and diseases: Expression of silvering 0not tested/1 absent (tol. to silvering)/8 present (susc. to silvering)Meloidogyne incognita 1 absent/9 present 9 Verticillium race 0 1absent/9 present 9 Fusarium oxysporum f. sp. lycopersici race 0 (old 1)1 absent/9 present 9 Fusarium oxysporum f. sp. lycopersici race 1 (old2) 1 absent/9 present 9 Fusarium oxysporium f. sp. radicis lycopersici 0not tested/1 absent/9 present. 1 Cladosporium fulvum group 0(Indeterminate varieties only) 0 not tested/1 absent/9 present. 1Cladosporium fulvum group A (Indeterminate varieties only) 1 absent/9present 1 Cladosporium fulvum group B (Indeterminate varieties only) 1absent/9 present 1 Cladosporium fulvum group C (Indeterminate varietiesonly) 1 absent/9 present 1 Cladosporium fulvum group D (Indeterminatevarieties only) 1 absent/9 present 1 Cladosporium fulvum group E(Indeterminate varieties only) 1 absent/9 present 1 Tomato mosaic virus(ToMV) strain 0 1 absent/9 present 9 Tomato mosaic virus (ToMV) strain 11 absent/9 present 9 Tomato mosaic virus (ToMV) strain 2 1 absent/9present 9 Tomato mosaic virus (ToMV) strain 1-2 0 not tested/1 absent/9present 9 Allele Tm 1 Tm1/2 Tm2/3 Tm2² 3 Special conditions for theexamination of the variety Type of culture: 1 glasshouse/2 outdoor/3other . . . 1 Type of culture 1 staked/2 semi staked/3 not staked 1 Mainuse: 1 fresh market or garden/2 industrial processing 1 If 1 freshmarket or garden: 1 single/2 truss/3 1 other, . . .

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed tomato plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the tomato plant(s).

Expression Vectors for Tomato Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983), Aragao F. J. L., et al., Molecular Breeding 4:6491-499 (1998). Another commonly used selectable marker gene is thehygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol.7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988), Saker M. M., etal, Biologia Plantarum 40:4 507-514 (1998), Russel, D. R., et al, PlantCell Report 12:3 165-169 (1993).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include beta-glucuronidase (GUS), alpha-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984), Grossi M. F., et al., Plant Science 103:2189-198 (1994), Lewis M. E., Journal of the American Society forHorticultural Science 119:2 361-366 (1994), Zhang et al., Journal of theAmerican Society for Horticultural Science 122:3 300-305 (1997).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green_, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression intomato. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in tomato. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression intomato or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in tomato.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985), Aragao et al., Genetics andMolecular Biology 22:3, 445-449 (1999) and the promoters from such genesas rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300(1992)).

The ALS promoter, XbaI/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said XbaI/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin tomato. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in tomato. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zml3(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981). According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is tomato. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syingae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btdelta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung tomato calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al.,Bio/Technology 10:1436 (1992). The cloning and characterization of agene which encodes a tomato endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to a Herbicide, for Example

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. See also Russel, D. R., et al, Plant Cell Report12:3 165-169 (1993). The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Accl-S1, Accl-S2 and Accl-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as

A. Delayed and attenuated symptoms to Tomato Golden Mosaic Geminivirus(BGMV), for example by transforming a plant with antisense genes fromthe Brazilian BGMV. See Arago et al., Molecular Breeding. 1998, 4: 6,491-499.

B. Increased the tomato content in Methionine by introducing a transgenecoding for a Methionine rich storage albumin (2S-albumin) from theBrazil nut as described in Arago et al., Genetics and Molecular Biology.1999, 22: 3, 445-449.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). McClean, P., et al. Plant CellTissue Org. Cult. 24(2, February), 131-138 (1991), Lewis et al., Journalof the American Society for Horticultural Science, 119:2, 361-366(1994), Zhang, Z., et al. J. Amer. Soc. Hort. Sci. 122(3): 300-305(1997). A. tumefaciens and A. rhizogenes are plant pathogenic soilbacteria which genetically transform plant cells. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal or vegetable crop species andgymnosperms have generally been recalcitrant to this mode of genetransfer, even though some success has recently been achieved in riceand corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat.No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 im. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993), Aragao, F. J. L.,et al. Plant Mol. Biol. 20(2, October), 357-359 (1992), Aragao Theor.Appl. Genet. 93:142-150 (1996), Kim, J.; Minamikawa, T. Plant Science117: 131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992)

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draperetal., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994).

Following transformation of tomato target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art. Theforegoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic tomato line. Alternatively, a genetic trait which hasbeen engineered into a particular tomato cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

When the term garden tomato plant, cultivar or tomato line is used inthe context of the present invention, this also includes any single geneconversions of that cultivar or line. The term single gene convertedplant as used herein refers to those garden tomato plants which aredeveloped by a plant breeding technique called backcrossing whereinessentially all of the desired morphological and physiologicalcharacteristics of a cultivar are recovered in addition to the singlegene transferred into the line via the backcrossing technique.Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the line. The term backcrossing asused herein refers to the repeated crossing of a hybrid progeny back toone of the parental tomato plants for that line. The parental tomatoplant which contributes the gene for the desired characteristic istermed the nonrecurrent or donor parent. This terminology refers to thefact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental tomato plant towhich the gene or genes from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In atypical backcross protocol, the original cultivar of interest (recurrentparent) is crossed to a second line (nonrecurrent parent) that carriesthe single gene of interest to be transferred. The resulting progenyfrom this cross are then crossed again to the recurrent parent and theprocess is repeated until a garden tomato plant is obtained whereinessentially all of the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred gene from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,herbicide resistance (such as bar or pat genes), resistance forbacterial, fungal, or viral disease such as gene I used for BCMVresistance), insect resistance, enhanced nutritional quality (such as 2salbumine gene), industrial usage, agronomic qualities such as the“persistent green gene”, yield stability and yield enhancement. Thesegenes are generally inherited through the nucleus. Some other singlegene traits are described in U.S. Pat. Nos. 5,777,196; 5,948,957 and5,969,212, the disclosures of which are specifically hereby incorporatedby reference.

All references cited herein are incorporated by reference in theapplication in their entireties.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somaclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

DEPOSIT

Applicants have made a deposit of at least 2500 seeds of inbred tomatolines TZ367 and TZ368, and tomato hybrid SX 387 with the American TypeCulture Collection (ATCC), Manassas, Va., 20110-2209 U.S.A., ATCCDeposit Nos: PTA-10147, PTA-10148 and PTA-10088, respectively. Thesedeposits will be maintained in the ATCC depository, which is a publicdepository, for a period of 30 years, or 5 years after the most recentrequest, or for the effective life of the patent, whichever is longer,and will be replaced if it becomes nonviable during that period.Additionally, Applicants have satisfied all the requirements of 37C.F.R. §§1.801-1.809, including providing an indication of the viabilityof the sample. Applicants impose no restrictions on the availability ofthe deposited material from the ATCC; however, Applicants have noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicants do notwaive any infringement of its rights granted under this patent or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

1. Seed of inbred tomato line TZ368, representative seed of said tomatoline having been deposited under ATCC Accession No. PTA-10148.
 2. Atomato plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. Pollen or an ovule of the plant of claim
 2. 4. A fruit ofthe plant of claim
 2. 5. A tissue culture of regenerable cells of aplant of inbred tomato line TZ368, wherein the tissue regenerates plantshaving all the morphological and physiological characteristics of aplant of inbred tomato line TZ368, representative seeds having beendeposited under ATCC Accession No. PTA-10148.
 6. The tissue culture ofclaim 5, selected from the group consisting of protoplast and calli,wherein the regenerable cells are produced from meristematic cells,leaves, pollen, embryo, root, root tips, stems, anther, flowers, seeds.7. A tomato plant regenerated from the tissue culture of claim 5,wherein the regenerated plant has all the morphological andphysiological characteristics of a plant of inbred tomato line TZ368,representative seeds having been deposited under ATCC Accession No.PTA-10148.
 8. A method for producing a hybrid tomato seed comprisingcrossing a first parent tomato plant with a second parent tomato plantand harvesting the resultant hybrid tomato seed, wherein said first orsecond parent tomato plant is the tomato plant of claim
 2. 9. A methodof producing an herbicide resistant tomato plant, an insect resistanttomato plant or a disease resistant tomato plant comprising transformingthe tomato plant of claim 2 with a transgene that confers herbicideresistance, insect resistance or resistance to bacterial, fungal orviral disease.
 10. An herbicide resistant tomato plant produced by themethod of claim
 9. 11. The tomato plant of claim 10, wherein thetransgene confers resistance to an herbicide selected from the groupconsisting of imidazolinone, sulfonylurea glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 12. An insect resistanttomato plant produced by the method of claim
 9. 13. The tomato plant ofclaim 12, wherein the transgene encodes a Bacillus thuringiensisprotein.
 14. A disease resistant tomato plant produced by the method ofclaim
 9. 15. A method of producing a tomato fruit comprising: a) growingthe tomato plant of claim 2 to produce a tomato fruit, and b) harvestingsaid tomato fruit.
 16. The method according to claim 15, furthercomprising packing said tomato fruit in a container.
 17. A method ofproducing a tomato seed comprising: a) growing the tomato plant of claim2 to produce a tomato seed, and b) harvesting said tomato seed.